Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

How DNA Copying Enzyme "Stops the Presses" for Repair Synthesizing Enzyme

31.03.2004


Lorena S. Beese, Ph.D.
PHOTO CREDIT: Duke University Medical Center


Biochemists have performed detailed structural studies that reveal for the first time how an enzyme key to DNA replication stalls when an error occurs, to allow it to be corrected. Without such instantaneous braking, such mistakes in DNA replication would wreak havoc on DNA replication, killing the cell.

To their surprise, the scientists observed how the enzyme, DNA polymerase, retains a "short-term memory" of mismatches, in some cases halting itself past the point of the mismatch, so that the repair machinery can go to work. They also found that the mismatch structures differed dramatically from those deduced from previous indirect biochemical studies.

In an article in the March 19, 2004, issue of the journal Cell, Duke University Medical Center biochemists Sean Johnson and Lorena Beese, Ph.D., described how they had conducted detailed structural analyses of DNA polymerase as it encountered each of the 12 possible kinds of mismatches possible in DNA replication.



In such replication, the polymerase sequentially attaches DNA units called bases along a single-stranded template DNA. The result is like constructing one rail of a spiral staircase, using the other rail as a guide; and the polymerase "translocates" the template strand through its active site like a thread through the eye of a needle.

In this replication process, the polymerase normally guides the template strand and assembles the complementary, growing "primer" strand by pairing each base with the correct counterpart -- always pairing adenine with thymine and cytosine with guanine.

When mismatches occur, the polymerase must instantly halt itself, triggering the mismatch repair machinery to launch into action, before replication can continue. This stalling is thought to occur because the polymerase-DNA structure is distorted by the mismatched bases, causing it to shut down.

The problem, said Beese, who is an associate professor of biochemistry, is that the critical molecular details of how such distortion acted to brake the polymerase have remained unknown.

"For 40 years, there have been biochemical studies trying to understand how polymerase achieves such a high fidelity of replication," said Beese. "It was known that the polymerase stalled, but it wasn’t known why. However, these studies represent the first direct observation of the structural details of mismatches and how they interact with the polymerase. And they show why and how stalling occurs."

In their studies, Beese and graduate student Johnson used the analytical technique of X-ray crystallography. In this widely used technique, intense X-ray beams are directed through a crystal of a protein to be analyzed, and the pattern of diffractions analyzed to deduce the structure of the protein.

The first steps in their studies were to first crystallize the polymerase with a segment of DNA containing each type of mismatched pair of nucleotides. Importantly, said Beese, the loosely associated crystals were so constructed that the polymerase could actually carry out several replication steps within the crystal.

"We have been able to replicate and translocate up to six base pairs in the crystal -- to my knowledge the biggest such motion ever seen in a crystal," said Beese. Using this approach, the Duke biochemists engineered the polymerase to be error-prone, so that they could produce crystals with mispaired bases inserted in the active site.

What’s more, they were able to move the mismatch away from the active site and still detect the distortion of the polymerase structure that would indicate the polymerase was "sensing" a mismatch. Thus, the enzyme could "remember" a mismatch after it had occurred.

"What was surprising about this finding is that prior to the study a mismatch was thought to induce only very small local distortions right around the mismatch," said Beese. "But what we saw is that the polymerase amplifies this distortion back to the active site." However, cautioned, Beese, the full details of the stalling mechanism under all possible conditions remain to be worked out. So, there could be other details of the stalling mechanism that could affect understanding of this "memory," she said.

Significantly, said Beese, she and Johnson discovered that both the growing primer strand and the template are involved in the stalling process.

"Although each mismatch is different, we saw that it isn’t just on the primer side that the structure is disrupted by a mismatch, but also on the template side, and sometimes both. And we also saw a mechanism we hadn’t expected at all, which is that some mismatches just get stuck and don’t translocate."

Although Beese emphasized that their studies are quite basic, such findings could help explain how the polymerase-triggered repair system is affected by DNA damage from carcinogenic chemicals.

The next steps in their research, said Beese, will be to instantaneously capture the polymerase in the act of processing a mismatch. The researchers plan to use flashes of ultraviolet light to unleash "caged" chemicals that trigger replication -- and at the same time use flashes of X-rays to illuminate the crystal. This approach may allow the researchers to make a movie of the polymerase during the synthesis and mismatch detection process.

Dennis Meredith | dukemed news
Further information:
http://dukemednews.org/news/article.php?id=7499

More articles from Life Sciences:

nachricht Monitoring biodiversity with sound: how machines can enrich our knowledge
18.06.2019 | Georg-August-Universität Göttingen

nachricht Uncovering hidden protein structures
18.06.2019 | Universität Konstanz

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: The hidden structure of the periodic system

The well-known representation of chemical elements is just one example of how objects can be arranged and classified

The periodic table of elements that most chemistry books depict is only one special case. This tabular overview of the chemical elements, which goes back to...

Im Focus: MPSD team discovers light-induced ferroelectricity in strontium titanate

Light can be used not only to measure materials’ properties, but also to change them. Especially interesting are those cases in which the function of a material can be modified, such as its ability to conduct electricity or to store information in its magnetic state. A team led by Andrea Cavalleri from the Max Planck Institute for the Structure and Dynamics of Matter in Hamburg used terahertz frequency light pulses to transform a non-ferroelectric material into a ferroelectric one.

Ferroelectricity is a state in which the constituent lattice “looks” in one specific direction, forming a macroscopic electrical polarisation. The ability to...

Im Focus: Determining the Earth’s gravity field more accurately than ever before

Researchers at TU Graz calculate the most accurate gravity field determination of the Earth using 1.16 billion satellite measurements. This yields valuable knowledge for climate research.

The Earth’s gravity fluctuates from place to place. Geodesists use this phenomenon to observe geodynamic and climatological processes. Using...

Im Focus: Tube anemone has the largest animal mitochondrial genome ever sequenced

Discovery by Brazilian and US researchers could change the classification of two species, which appear more akin to jellyfish than was thought.

The tube anemone Isarachnanthus nocturnus is only 15 cm long but has the largest mitochondrial genome of any animal sequenced to date, with 80,923 base pairs....

Im Focus: Tiny light box opens new doors into the nanoworld

Researchers at Chalmers University of Technology, Sweden, have discovered a completely new way of capturing, amplifying and linking light to matter at the nanolevel. Using a tiny box, built from stacked atomically thin material, they have succeeded in creating a type of feedback loop in which light and matter become one. The discovery, which was recently published in Nature Nanotechnology, opens up new possibilities in the world of nanophotonics.

Photonics is concerned with various means of using light. Fibre-optic communication is an example of photonics, as is the technology behind photodetectors and...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

SEMANTiCS 2019 brings together industry leaders and data scientists in Karlsruhe

29.04.2019 | Event News

Revered mathematicians and computer scientists converge with 200 young researchers in Heidelberg!

17.04.2019 | Event News

First dust conference in the Central Asian part of the earth’s dust belt

15.04.2019 | Event News

 
Latest News

Uncovering hidden protein structures

18.06.2019 | Life Sciences

Monitoring biodiversity with sound: how machines can enrich our knowledge

18.06.2019 | Life Sciences

Schizophrenia: Adolescence is the game-changer

18.06.2019 | Life Sciences

VideoLinks
Science & Research
Overview of more VideoLinks >>>